Treatment for atherosclerosis and other cardiovascular and inflammatory diseases

ABSTRACT

A method for the treatment of cardiovascular diseases and noncardiovascular inflammatory diseases that are mediated by VCAM-1 is provided that includes the removal, decrease in the concentration of, or prevention of the formation of oxidized polyunsaturated fatty acids, or interferes with a complex formed between a polyunsaturated fatty acid or an oxidized polyunsaturated fatty acid and a protein or peptide that mediates the expression of VCAM-1. A method is also provided for suppressing the expression of a redox-sensitive gene or activating a gene that is suppressed through a redox-sensitive pathway, that includes administering an effective amount of a substance that prevents the oxidation of the oxidized signal, and typically, the oxidation of a polyunsaturated fatty acid, or interferes with a complex formed between the oxidized signal and a protein or peptide that mediates the expression of the redox gene.

BACKGROUND OF THE INVENTION

The U.S. government may have rights in this invention by virtue of agrant from the National Institutes of Health that partially funded workleading to the invention.

This application is in the area of methods and compositions for thetreatment of atherosclerosis and other cardiovascular and inflammatorydiseases.

This application is a continuation-in-part of U.S. Ser. No. 08/240,858,now abandoned, filed on May 10, 1994 by Russell M. Medford, Margaret K.Offermann, Wayne R. Alexander, and Sampath Parthasarathy entitled"Treatment of Atherosclerosis and Other Cardiovascular and InflammatoryDiseases," which is a continuation-in-part of U.S. Ser. No. 07/969,934,now U.S. Pat. No. 5,380,747 filed on Oct. 30, 1992 by Russell M.Medford, Margaret K. Offermann, and R. Wayne Alexander, entitled"Treatment of Atherosclerosis and Other Cardiovascular and InflammatoryDiseases," now allowed.

Adhesion of leukocytes to the endothelium represents a fundamental,early event in a wide variety of inflammatory conditions, includingatherosclerosis, autoimmune disorders and bacterial and viralinfections. Leukocyte recruitment to the endothelium is started wheninducible adhesion molecule receptors on the surface of endothelialcells interact with counterreceptors on immune cells. Vascularendothelial cells determine which type of leukocytes (monocytes,lymphocytes, or neutrophils) are recruited, by selectively expressingspecific adhesion molecules, such as vascular cell adhesion molecule-1(VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and E-selectin. Inthe earliest stage of the atherosclerotic lesion, there is a localizedendothelial expression of VCAM-1 and selective recruitment ofmononuclear leukocytes that express the integrin counterreceptor VLA-4.Because of the selective expression of VLA-4 on monocytes andlymphocytes, but not neutrophils, VCAM-1 is important in mediating theselective adhesion of mononuclear leukocytes. Subsequent conversion ofleucocytes to foamy macrophages results in the synthesis of a widevariety of inflammatory cytokines, growth factors, and chemoattractantsthat help propagate the leukocyte and platelet recruitment, smoothmuscle cell proliferation, endothelial cell activation, andextracellular matrix synthesis characteristic of maturingatherosclerotic plaque.

VCAM-1 is expressed in cultured human vascular endothelial cells afteractivation by lipopolysaccharide (LPS) and cytokines such asinterleukin-1 (IL-1) and tumor necrosis factor (TNF-α). These factorsare not selective for activation of cell adhesion molecule expression.

Molecular analysis of the regulatory elements on the human VCAM-1 genethat control its expression suggests an important role for nuclearfactor-kB (NF-kB), a transcriptional regulatory factor, or an NF-kβ likebinding protein in oxidation-reduction-sensitive regulation of VCAM-1gene expression. Transcriptional factors are proteins that activate (orrepress) gene expression within the cell nucleus by binding to specificDNA sequences called "enhancer elements" that are generally near theregion of the gene, called the "promoter," from which RNA synthesis isinitiated. Nuclear factor-kB is a ubiquitously expressed multisubunittranscription factor activated in several cell types by a large anddiverse group of inflammatory agents such as TNFα, IL-1β, bacterialendotoxin, and RNA viruses. It plays a key role in mediatinginflammatory and other stress signals to the nuclear regulatoryapparatus. Although the precise biochemical signals that activate NF-kBare unknown, this transcriptional factor may integrate into a commonmolecular pathway many of the risk factors and "causative" signals ofatherosclerosis, such as hyperlipidemia, smoking, hypertension, anddiabetes mellitus.

Importantly, the activation of NF-kB in vascular endothelial cells bydiverse signals can be specifically inhibited by antioxidants such asN-acetylcysteine and pyrrolidine dithiocarbamate (see U.S. Ser. No.07/969,934, now allowed). This has led to the hypothesis that oxygenradicals play an important role in the activation of NF-kB through anundefined oxidation-reduction mechanism. Because an NF-kB-like enhancerelement also regulates the transcription of the VCAM-1 promoter in anoxidation-reduction-sensitive manner, oxidative stress in theatherosclerotic lesion may play a role in regulating VCAM-1 geneexpression through this oxidation-reduction-sensitive transcriptionalregulatory protein.

It has been hypothesized that modification of low-density lipoprotein(LDL) into oxidatively modified LDL (ox-LDL) by reactive oxygen speciesis the central event that initiates and propagates atherosclerosis.Steinberg, et al., N. Engl. J. Med. 1989; 320:915-924. Oxidized LDL is acomplex structure consisting of at least several chemically distinctoxidized materials, each of which, alone or in combination, may modulatecytokine-activated adhesion molecule gene expression. Fatty acidhydroperoxides such as linoleyl hydroperoxide (13-HPODE) are producedfrom free fatty acids by lipoxygenases and are an important component ofoxidized LDL.

It has been proposed that a generation of oxidized lipids is formed bythe action of the cell lipoxygenase system and that the oxidized lipidsare subsequently transferred to LDL. There is thereafter a propagationreaction within the LDL in the medium catalyzed by transition metalsand/or sulfhydryl compounds Previous investigations have demonstratedthat fatty acid modification of cultured endothelial cells can altertheir susceptibility to oxidant injury. Supplementation of saturated ormonounsaturated fatty acids to cultured endothelial cells reduces theirsusceptibility to oxidant injury, whereas supplementation withpolyunsaturated fatty acids (PUFA) enhances susceptibility to oxidantinjury.

Using reverse-phase HPLC analysis of native and saponified lipidextracts of LDL, it has been demonstrated that 13-HPODE is thepredominant oxidized fatty acid in LDL oxidized by activated humanmonocytes. Chronic exposure to oxidized LDL provides an oxidative signalto vascular endothelial cells, possibly through a specific fatty acidhydroperoxide, that selectively augments cytokine-induced VCAM-1 geneexpression.

Through a mechanism that is not well defined, areas of vessel wallpredisposed to atherosclerosis preferentially sequester circulating LDL.Through a poorly understood pathway, endothelial, smooth muscle, and/orinflammatory cells then convert LDL to ox-LDL. In contrast to LDL, whichis taken up through the LDL receptor, monocytes avidly take up ox-LDLthrough a "scavenger" receptor whose expression, unlike the LDLreceptor, is not inhibited as the content of intracellular lipid rises.Thus, monocytes continue to take up ox-LDL and become lipid-engorgedmacrophage-foam cells that form the fatty streak.

Given that cardiovascular disease is currently the leading cause ofdeath in the United States, and ninety percent of cardiovascular diseaseis presently diagnosed as atherosclerosis, there is a strong need toidentify new methods and pharmaceutical agents for its treatment.Important to this goal is the identification and manipulation of thespecific oxidized biological compounds that act as selective regulatorsof the expression of mediators of the inflammatory process, and inparticular, VCAM-1. A more general goal is to identify selective methodsfor suppressing the expression of redox sensitive genes or activatingredox sensitive genes that are suppressed.

It is therefore an object of the present invention to provide atreatment for atherosclerosis and other cardiovascular and inflammatorydiseases.

It is another object of the present invention to provide a method forthe selective inhibition of VCAM-1.

It is still another object of the present invention to provide a methodfor the treatment of a human disease or disorder that is mediated by theexpression or suppression of a redox sensitive gene.

It is another object of the present invention to provide pharmaceuticalcompositions for the treatment of atherosclerosis and othercardiovascular and inflammatory diseases.

SUMMARY OF THE INVENTION

It has been discovered that polyunsaturated fatty acids ("PUFAs") andtheir hydroperoxides ("ox-PUFAs"), which are important components ofoxidatively modified low density lipoprotein (LDL), induce theexpression of VCAM-1, but not intracellular adhesion molecule-1 (ICAM-1)or E-selectin in human aortic endothelial cells, through a mechanismthat is not mediated by cytokines or other noncytokine signals. This isa fundamental discovery of a an important and previously unknownbiological pathway in VCAM-1 mediated immune responses.

As nonlimiting examples, linoleic acid, linolenic acid, arachidonicacid, linoleyl hydroperoxide (13-HPODE) and arachidonic hydroperoxide(15-HPETE) induce cell-surface gene expression of VCAM-1 but not ICAM-1or E-selectin. Saturated fatty acids (such as stearic acid) andmonounsaturated fatty acids (such as oleic acid) do not induce theexpression of VCAM-1, ICAM-1, or E-selectin.

The induction of VCAM-1 by PUFAs and their fatty acid hydroperoxides issuppressed by the antioxidant pyrrolidine dithiocarbamate (PDTC). Thisindicates that the induction is mediated by an oxidized signal molecule,and that the induction is prevented when the oxidation of the moleculeis blocked (i.e., the oxidation does not occur), reversed (i.e., thesignal molecule is reduced), or when the redox modified signal isotherwise prevented from interacting with its regulatory target.

Cells that are chronically exposed to higher than normal levels ofpolyunsaturated fatty acids or their oxidized counterparts can initiatean immune response that is not normal and which is out of proportion tothe threat presented, leading to a diseased state. The oversensitizationof vascular endothelial cells to PUFAS and ox-PUFAS can accelerate theformation, for example, of atherosclerotic plaque.

Based on these discoveries, a method for the treatment ofatherosclerosis, post-angioplasty restenosis, coronary artery diseases,angina, and other cardiovascular diseases, as well as noncardiovascularinflammatory diseases that are mediated by VCAM-1, is provided thatincludes the removal, decrease in the concentration of, or prevention ofthe formation of oxidized polyunsaturated fatty acids including but notlimited to oxidized linoleic (C₁₈ Δ⁹,12) , linolenic (C₁₈ Δ⁶,9,12) ,arachidonic (C₂₀ Δ⁵,8,11,14) and eicosatrienoic (C₂₀ Δ⁸,11,14) acids.Nonlimiting examples of noncardiovascular inflammatory diseases that aremediated by VCAM-1 include rheumatoid and osteoarthritis, asthma,dermatitis, and multiple sclerosis.

This method represents a significant advance in treating cardiovasculardisease, in that it goes beyond the current therapies designed simply toinhibit the progression of the disease, and when used appropriately,provides the possibility to medically "cure" atherosclerosis bypreventing new lesions from developing and causing established lesionsto regress.

In an alternative embodiment, a method is provided for suppressing theexpression of a redox-sensitive gene or activating a gene that issuppressed through a redox-sensitive pathway, that includesadministering an effective amount of a substance that prevents theoxidation of the oxidized signal, and typically, the oxidation of apolyunsaturated fatty acid. Representative redox-sensitive genes thatare involved in the presentation of an immune response include, but arenot limited to, those expressing cytokines involved in initiating theimmune response (e.g., IL-1β) , chemoattractants that promote themigration of inflammatory cells to a point of injury (e.g., MCP-1),growth factors (e.g., IL-6 and the thrombin receptor), and adhesionmolecules (e.g., VCAM-1 and E-selectin).

Screens for disorders mediated by VCAM-1 or a redox-sensitive gene arealso provided that include the quantification of surrogate markers ofthe disease. In one embodiment, the level of oxidized polyunsaturatedfatty acid, or other appropriate markers, in the tissue or blood, forexample, of a host is evaluated as a means of assessing the "oxidativeenvironment" of the host and the host's susceptibility to VCAM-1 orredox-sensitive gene mediated disease.

In another embodiment, the level of circulating or cell-surface VCAM-1or other appropriate marker and the effect on that level ofadministration of an appropriate antioxidant is quantified.

In yet another assay, the sensitization of a host's vascular endothelialcells to polyunsaturated fatty acids or their oxidized counterparts isevaluated. This can be accomplished, for example, by challenging a hostwith a PUFA or ox-PUFA and comparing the resulting concentration ofcell-surface or circulating VCAM-1 or other surrogate marker to apopulation norm.

In another embodiment, in vivo models of atherosclerosis or other heartor inflammatory diseases that are mediated by VCAM-1 can be provided byadministering to a host animal an excessive amount of PUFA or oxidizedpolyunsaturated fatty acid to induce disease. These animals can be usedin clinical research to further the understanding of these disorders.

In yet another embodiment of the invention, compounds can be assessedfor their ability to treat disorders mediated by VCAM-1 on the basis oftheir ability to inhibit the oxidation of a polyunsaturated fatty acid,or the interaction of a PUFA or ox-PUFA with a protein target.

This can be accomplished by challenging a host, for example, a human oran animal such as a mouse, to a high level of PUFA or ox-PUFA and thendetermining the therapeutic efficacy of a test compound based on itsability to decrease circulating or cell surface VCAM-1 concentration.Alternatively, an in vitro screen can be used that is based on theability of the test compound to prevent the oxidation of a PUFA, or theinteraction of a PUFA or ox-PUFA with a protein target in the presenceof an oxidizing substance such as a metal, for example, copper, or anenzyme such as a peroxidase, lipoxygenase, cyclooxygenase, or cytochromeP450.

In another embodiment, vascular endothelial cells are exposed to TNF-αor other VCAM-1 inducing material for an appropriate time and thenbroken by any appropriate means, for example by sonication orfreeze-thaw. The cytosolic and membrane compartments are isolated.Radiolabeled PUFA is added to defined amounts of the compartments. Theability of the liquid to convert PUFA to ox-PUFA in the presence orabsence of a test compound is assayed. Intact cells can be used in placeof the broken cell system.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of the cell-surface expression (O.D. 450 nm) of VCAM-1as a function of hours in human aortic endothelial cells on exposure tothe cytokine TNF-α (closed circle); linoleic acid (closed triangle); andlinoleyl hydroperoxide (13-HPODE, closed square); and in the absence ofexposure to these substances (control, open square).

FIG. 2 is a graph of the cell-surface expression (O.D. 450 nm) of VCAM-1in human aortic endothelial cells on exposure to linoleic acid (closedtriangle) and linoleyl hydroperoxide (13-HPODE, closed square) as afunction of the concentration of fatty acid (μM).

FIG. 3 is a bar chart graph of the cell-surface expression (O.D. 450 nm)of VCAM-1, ICAM-1 and E-selectin in human aortic endothelial cells onexposure to the cytokine TNF-α, stearic acid, oleic acid, linoleic acid,linolenic acid, and arachidonic acid.

FIG. 4 is a bar chart graph of the cell-surface expression (O.D. 450 nm)of VCAM-1 in human aortic endothelial cells on exposure to linoleicacid, 13-HPODE, arachidonic acid, and arachidonic acid hydroperoxide(15-HPETE), with (solid black) or without (hatched lines) theantioxidant pyrrolidine dithiocarbamate.

FIG. 5 is an illustration of an autoradiogram indicating the acuteinduction of VCAM-1 MRNA by linoleic acid and 13-HPODE. HAEC wereexposed or not to linoleic acid (7.5 μM), 13-HPODE (7.5 μM) or TNF-α(100U/ml). Total RNA was isolated and 20 μg was size-fractionated bydenaturing 1.0% agarose-formaldehyde gel electrophoresis, transferred tonitrocellulose, and hybridized to either ³² P-labeled human A) VCAM-1specific or B) β-actin specific cDNA. After washing, the filters wereexposed to X-ray film at -70° C. with one intensifying screen for 24hours. Identification of lanes: 1) control; 2) linoleic acid (acute,8-hour exposure); 3) linoleic acid (48-hour exposure); 4) 13-HPODE(acute, 8-hour exposure); and 5) TNF-α(100 U/ml, 4-hour exposure).

FIG. 6 is an illustration of an autoradiogram that indicates thatinduction of VCAM-1 mRNA by polyunsaturated fatty acids is independentof cellular protein synthesis. HAEC were exposed to either linoleic (7.5μM) or arachidonic (7.5 μM) acid in the presence or absence ofcycloheximide (10 μg/ml) for a 4-hour period, and then treated asdescribed in FIG. 5.

FIG. 7 is an illustration of an autoradiogram that indicates thatlinoleic acid induces transcriptional activation of the VCAM-1 promoterby a redox-sensitive NF-kB like factor. HAEC were split at the ratio togive approximately 60% confluence in 100-mm tissue culture plates. HAECwere transfected with either 30 μg of p288 VCAMCAT, p85 VCAMCAT, or pSV₂CAT plasmid by the calcium phosphate coprecipitation technique usingstandard techniques. After a 24-hour recovery period, HAEC werepretreated or not with 50 μM PDTC and after 30 minutes exposed tolinoleic acid (7.5 μM) or TNF-α (100 U/ml) directly added to the plates.After 18 hours, cell extracts were prepared by rapid freeze-thaw in 0.25M Tris, pH 8.0. The protein of each cell extract was assayed forchloramphenicol acetyl transferase (CAT) activity, as previouslydescribed Ausubel, 1989! (Ac, acetylated; N, nonacetylatedchloramphenicol).

FIG. 8 is an illustration of an acrylamide gel slab that indicates thatpolyunsaturated fatty acids activate NF-kB-like DNA binding activitiesthat are blocked by the antioxidant PDTC. Confluent HAEC in mediacontaining 4% FBS (as described in FIG. 1) were pretreated or not withPDTC (50 μM) for thirty minutes and then exposed for three hours tolinoleic acid (7.5 μM, oleic acid (7.5 μM), or TNFα (100 U/ml),respectively. Five micrograms of nuclear extract was incubated with adouble-stranded ³² P-labeled wtVCAM, size fractionated on 4% nativeacrylamide gels, and exposed to autoradiography film at -70° C. for 18hours. Two bands A and C, representing NF-kB like binding activity aredesignated. A weak band B was observed in control (untreated) cells.

FIGS. 9A and 9B are bar chart graphs of the relative thiabarbituric acidreactive substances (O.D. 532 nm) of arachidonic acid and 15-HPETE inthe presence or absence of PDTC. The thiobarbituric acid reactivityassay (TBARS) measures the oxidation ability of a material in acell-free, media-free environment.

FIG. 10 is an illustration of an autoradiogram of MRNA, obtained asdescribed below, hybridized to either 32P-labeled human VCAM-1 specificcDNA (Panel A), E-selectin (ELAM-1) specific cDNA (Panel B), or ICAM-1specific cDNA (Panel C). Following pre-treatment for 30 minutes with 50μM of sodium pyrrolidine dithiocarbamate (PDTC), HUVE (human umbilicalvein) cells were exposed to IL-lb (10 U/ml) in the continuous presenceof 50 μM PDTC. Parallel controls were performed identically except inthe absence of PDTC. At the indicated times, total RNA was isolated and20 μg of material size-fractionated by denaturing 1.0%agarose-formaldehyde gel electrophoresis, transferred to nitrocellulose,hybridized as described above, and visualized by autoradiography. Lane1-0 hour; Lanes 2,4,6,8 -OL-1 alone for 2, 4, 8 and 24 hours,respectively; Lanes 3,5,7,9 - IL-1 with PDTC for 2,4,8 and 24 hours,respectively.

FIG. 11 is an illustration of an autoradiogram of mRNA, obtained asdescribed below, hybridized to either 32P-labeled human VCAM-1 specific(Panel A), E-selectin (ELAM-1) specific CDNA (Panel B), or ICAM-1specific cDNA (Panel C). HUVE cells were pretreated with the indicatedconcentrations of PDTC, and then exposed to IL-lb in the presence ofPDTC for four hours and assayed for VCAM-1 mRNA accumulation by Northernfilter hybridization analysis. Lane 1--control, lane 2--IL-1 (10 u/ml),lane 3--IL-lb+PDTC (0.05 μM), lane 4--IL-1 LB +PDTC (0.5 μM), lane5--IL-lb+PDTC (5.0 μM), lane 6--IL=1b+PDTC (50.0 μM), lane 7--IL-lb+PDTC(100 μM).

FIG. 12 is an illustration of an autoradiogram of mRNA, obtained asdescribed below, hybridized to either 32P-labeled human VCAM-1 specificcDNA (Panel A), E-selectin (ELAM-1) specific cDNA (Panel B), or ICAM-1specific cDNA (Panel C). HUVE cells were pretreated as described in FIG.9 with 50 μM PDTC, exposed for four hours to the agents indicated below,and assayed for VCAM-1 (Panel A) and ICAM-1 (Panel B) mRNA accumulation.Lane 1--TNFa (100 U/ml), lane 2--TNFa+PDTC, lane 3--lipopolysaccharide(LPS) (100 ng/ml), lane 4--LPS+PDTC, lane 5 --poly(I:C) (100 mg/ml),lane 6--poly(I:C)+PDTC.

FIG. 13 is a graph of relative cell surface expression of VCAM-1 andICAM-1 in the presence (dark bars) or absence (white bars) of PDTC andin the presence of multiple types of inducing stimuli. Confluent HUVECswere pretreated or not pretreated (CTL only) for 30 minutes with 50 μMPDTC, and then exposed for the indicated times to the indicated agentsin the presence or absence (CTL only) of PDTC. Cell surface expressionwas determined by primary binding with VCAM-1 specific (4B9) and ICAM-1specific (84H10) mouse monoclonal antibodies followed by secondarybinding with a horse-radish peroxidase tagged goat anti-mouse (IgG).Quantitation was performed by determination of calorimetric conversionat 450 nm of TMB. FIG. 13 indicates that multiple regulatory signalsinduce VCAM-1 but not ICAM-1 through a common, dithiocarbamate-sensitivepathway in human vascular endothelial cells.

FIG. 14 is a graph of the relative VCAM-1 cell surface expression (O.D.595 nM) in human umbilical vein endothelial cells, activated by TNFa,versus concentration of various antioxidants. (PDTC is sodiumN-pyrrolidine dithiocarbamate; DETC is sodiumN,N-diethyl-N-carbodithiolate, also referred to as sodiumdiethyldithiocarbamate; NAC is N-acetyl cysteine; and DF isdesferroximine).

FIG. 15 is a graph of the relative VCAM-1 cell surface expression (O.D.595 nM) in human umbilical vein endothelial cells, activated by TNF-α,in the presence of the specified amount of antioxidant. (PDTC is sodiumN-pyrrolidine dithiocarbamate; DIDTC is sodiumN,N-diethyl-N-carbodithioate; SarDTC is sodiumN-methyl-N-carboxymethyl-N-carbodithioate; IDADTC is trisodiumN,N-di(carboxymethyl)-N-carbodithioate; MGDTC is sodiumN-methyl-D-glucamine-N-carbodithioate; MeOBGDTC is sodiumN-(4-methoxybenzyl)-D-glucamine-N-carbodithioate; DEDTC is sodiumN,N-diethyl-N-carbodithioate; Di-PDTC is sodiumN,N-diisopropyl-N-carbodithioate; NAC is N-acetyl cysteine.)

FIG. 16 is a graph of the percentage of Molt-4 cells binding to HUVEcells either unstimulated or stimulated with TNFa (100 U/ml) for sixhours in the presence or absence of PDTC.

FIG. 17 is an illustration of the chemical structures of the followingactive dithiocarbamates: sodium pyrrolidine-N-carbodithioate, sodiumN-methyl-N-carboxymethyl-N-carbodithioate, trisodiumN,N-di(carboxymethyl)-N-carbodithioate, sodiumN-methyl-D-glucamine-N-carbodithioate, sodiumN,N-diethyl-N-carbodithioate (sodium diethyldithiocarbamate), and sodiumN,N-diisopropyl-N-carbodithioate.

FIG. 18 is a bar chart graph of the effect of PTDC on the formation offluorescent adducts of BSA and 13-HPODE, as measured in fluorescentunits versus micromolar concentration of PDTC. One micromolar of13-HPODE was incubated with 200 micrograms of BSA in the presence ofPDTC for six days. Fluorescence was measured at 430-460 nm withexcitation at 330-360 nm.

FIG. 19 is a graph of the effect of PTDC on the formation of fluorescentadducts of BSA and ox-PUFA as a function of wavelength (nm) andconcentration of PDTC. As the concentration of PDTC increases, thequantity of fluorescent adducts decrease.

FIG. 20 is a graph of the effect of PDTC on the oxidation of LDL byhorseradish peroxidase (HRP), as measured by the increase in O.D. (234nm) versus time (minutes) for varying concentrations of PDTC. It isobserved that after an incubation period, PDTC inhibits the oxidation ofLDL by HRP in a manner that is concentration dependent.

FIG. 21 is a chart of the effect of PDTC on the cytokine-inducedformation of ox-PUFA in human aortic endothelial cells. As indicated,both TNF-α and IL-1B causes the oxidation of linoleic acid toox-linoleic acid. The oxidation is significantly prevented by PDTC.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein, the term polyunsaturated fatty acid (also referred toherein as a "PUFA") refers to a fatty acid (typically C₈ to C₂₄) thathas at least two alkenyl bonds, and includes but is not limited tolinoleic (C₁₈ Δ⁹,12), linolenic (C₁₈ Δ⁶,9,12), arachidonic (C₂₀Δ⁵,8,11,14) and eicosatrienoic (C₂₀ Δ⁸,11,14) acids.

The term oxidized polyunsaturated fatty acid refers to an unsaturatedfatty acid in which at least one of the alkenyl bonds has been convertedto a hydroperoxide of the structure. Nonlimiting examples are: ##STR1##

The term alkyl, as used herein, unless otherwise specified, refers to asaturated straight, branched, or cyclic (in the case of C₅ or greater)hydrocarbon of C₁ to C₁₀ (or lower alkyl, i.e., C₁ to C₅), whichspecifically includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl,t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl,cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, and2,3-dimethylbutyl. The alkyl group can be optionally substituted on anyof the carbons with one or more moieties selected from the groupconsisting of hydroxyl, amino, or mono- or disubstituted amino, whereinthe substituent group is independently alkyl, aryl, alkaryl or aralkyl;aryl, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonicacid, phosphate, or phosphonate, either unprotected, or protected asnecessary, as known to those skilled in the art, for example, as taughtin Greene, et al., "Protective Groups in organic Synthesis," John Wileyand Sons, Second Edition, 1991.

The term alkenyl, as referred to herein, and unless otherwise specified,refers to a straight, branched, or cyclic hydrocarbon of C₂ to C₁₀ withat least one double bond.

The term alkynyl, as referred to herein, and unless otherwise specified,refers to a C₂ to C₁₀ straight or branched hydrocarbon with at least onetriple bond.

The term aralkyl refers to an aryl group with at least one alkylsubstituent.

The term alkaryl refers to an alkyl group that has at least one arylsubstituent.

The term halo (alkyl, alkenyl, or alkynyl) refers to an alkyl, alkenyl,or alkynyl group in which at least one of the hydrogens in the group hasbeen replaced with a halogen atom.

The term aryl, as used herein, and unless otherwise specified, refers tophenyl, biphenyl, or naphthyl, and preferably phenyl. The aryl group canbe optionally substituted with one or more moieties selected from thegroup consisting of alkyl, hydroxyl, amino, alkylamino, arylamino,alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid,phosphate, or phosphonate, CO₂ H, or its pharmaceutically acceptablesalt, CO₂ (alkyl, aryl, alkaryl or aralkyl), or glucamine, eitherunprotected, or protected as necessary, as known to those skilled in theart, for example, as taught in Greene, et al., "Protective Groups inOrganic Synthesis," John Wiley and Sons, Second Edition, 1991.

The term alkoxy, as used herein, and unless otherwise specified, refersto a moiety of the structure -O-alkyl.

The term acyl, as used herein, refers to a group of the formula C(O)R',wherein R' is an alkyl, aryl, alkaryl or aralkyl group.

The term heteroaryl or heteroaromatic, as used herein, refers to anaromatic moiety that includes at least one sulfur, oxygen, or nitrogenin the aromatic ring. Nonlimiting examples are phenazine, phenothiazine,furyl, pyridyl, pyrimidyl, thienyl, isothiazolyl, imidazolyl,tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl,isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, indolyl,isoindolyl, benzimidazolyl, purinyl, morpholinyl, carbozolyl, oxazolyl,thiazolyl, isothiazolyl, 1,2,4-thiadiazolyl, isooxazolyl, pyrrolyl,pyrazolyl, quinazolinyl, pyridazinyl, pyrazinyl, cinnolinyl,phthalazinyl, quinoxalinyl, xanthinyl, hypoxanthinyl, pteridinyl,5-azacytidinyl, 5-azauracilyl, triazolopyridinyl, imidazolopyridinyl,pyrrolopyrimidinyl, pyrazolopyrimidinyl, adenine, N⁶ -alkylpurines, N⁶-benzylpurine, N⁶ -halopurine, N⁶ -vinylpurine, N⁶ - acetylenic purine,N⁶ -acyl purine, N⁶ -hydroxyalkyl purine, N⁶ -thioalkyl purine, thymine,cytosine, 6-azapyrimidine, 2-mercaptopyrmidine, uracil, N⁵-alkylpyrimidines, N⁵ -benzylpyrimidines, N⁵ -halopyrimidines, N⁵-vinylpyrimidine, N⁵ -acetylenic pyrimidine, N⁵ -acyl pyrimidine, N⁵-hydroxyalkyl purine, and N⁶ -thioalkyl purine, and isoxazolyl. Theheteroaromatic group can be optionally substituted as described abovefor aryl. The heteroaromatic can be partially or totally hydrogenated asdesired. As a nonlimiting example, dihydropyridine can be used in placeof pyridine. Functional oxygen and nitrogen groups on the heterocyclicbase can be protected as necessary or desired during the reactionsequence. Suitable protecting groups are well known to those skilled inthe art, and include trimethylsilyl, dimethylhexylsilyl,t-butyldimethylsilyl, and t-butyldiphenylsilyl, tritylmethyl, alkylgroups, acyl groups such as acetyl and propionyl, methylsulfonyl, andp-toluylsulfonyl.

The term hydroxyalkyl, as used herein, refers to a C₁ to C₆ alkyl groupin which at least one of the hydrogens attached to any of the carbonatoms is replaced with a hydroxy group.

The term thiol antioxidant refers to a sulfur containing compound thatretards oxidation.

The term pharmaceutically acceptable derivative refers to a derivativeof the active compound that upon administration to the recipient, iscapable of providing directly or indirectly, the parent compound, orthat exhibits activity itself.

The term "pharmaceutically acceptable cation" refers to an organic orinorganic moiety that carries a positive charge and that can beadministered in association with a pharmaceutical agent, for example, asa countercation in a salt. Pharmaceutically acceptable cations are knownto those of skill in the art, and include but are not limited to sodium,potassium, and quaternary amine.

The term "physiologically cleavable leaving group" refers to a moietythat can be cleaved in vivo from the molecule to which it is attached,and includes but is not limited to an organic or inorganic anion, apharmaceutically acceptable cation, acyl (including but not limited to(alkyl)C(O), including acetyl, propionyl, and butyryl), alkyl,phosphate, sulfate and sulfonate.

The term "enantiomerically enriched composition or compound" refers to acomposition or compound that includes at least 95%, and preferably atleast 97, 98, 99, or 100% by weight of a single enantiomer of thecompound.

The term amino acid includes synthetic and naturally occurring aminoacids, including but not limited to, for example, alanyl, valinyl,leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl,methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl,asparaginyl, glutaminyl, aspartoyl, glutaoyl, lysinyl, argininyl, andhistidinyl.

A "linking moiety" as used herein, is any divalent group that links twochemical residues, including but not limited to alkyl, alkenyl, alkynyl,aryl, polyalkyleneoxy (for example -- (CH₂)_(n) O--!_(n) --), --C₁₋₆alkoxy-C₁₋₁₀ alkyl-, --C₁₋₆ alkylthio-C₁₋₁₀ alkyl-, --NR³ --, and--(CHOH_(n) CH₂ OH, wherein n is independently 0, 1, 2, 3, 4, 5, or 6.

II. Identification of Oxidized and Unoxidized Polyunsaturated FattyAcids as Direct Mediators of VCAM-1 Expression

To establish whether a PUFA or oxidized PUFA acts as a directimmunomodulator of endothelial cell gene expression, early passagedhuman aortic endothelial cells (HAEC) were cultured for eight hours inmedia and serum and exposed to saturated (stearic), monounsaturated(oleic), and polyunsaturated (linoleic and arachidonic) fatty acids; aswell as with the fatty acid hydroperoxides of linoleic (13-HPODE) orarachidonic (15-HPETE) acids. HAEC were also alternatively exposed tothe cytokine tumor necrosis factor-α.

HAEC were exposed to linoleic acid or 13-HPODE for varying times up to48 hours and then assayed for cell surface VCAM-1 expression by ELISAassay. The results were compared to HAEC exposed to the cytokine TNF-α(100 U/ml) for the same time periods. VCAM-1 expression in HAECincubated with either linoleic acid or 13-HPODE is transiently induced.The expression peaks at approximately 8-9 hours with significantexpression at 24 hours and then decreases by 48 hours. The kinetics ofVCAM-1 induction by both linoleic acid and 13-HPODE mirror that ofTNF-α, and thus the mechanisms by which polyunsaturated fatty acidsinduce VCAM-1 thus appear to be similar to that of TNF-α.

Dose-response studies of linoleic acid and 13-HPODE on VCAM-1 geneexpression at 8 hours were also conducted. It was observed that 7.5 μMis the lowest peak dose by which linoleic acid and 13-HPODE inducessignificant VCAM-1 gene expression.

It was then explored whether short term incubation of endothelial cellswith polyunsaturated fatty acids induces ICAM-1 and E-selectinexpression as well. It was determined that the polyunsaturated fattyacids linoleic and arachidonic acids induced cell-surface geneexpression to approximately 59% of TNF-induced gene expression ofVCAM-1. Strikingly, neither ICAM-1 nor E-selectin were induced by thesefatty acids. Conversely, the saturated fatty acid stearic acid and themonounsaturated fatty acid oleic acid did not induce the expression ofVCAM-1, ICAM-1, or E-selectin. VCAM-1 gene expression was also observedby incubation of HAEC with the oxidized metabolites of linoleic acid(13-HPODE) and arachidonic acid (15-HPETE).

To investigate whether oxidative stress in endothelial cells provided bypolyunsaturated fatty acids and their oxidized metabolites inducesVCAM-1 through a redox-sensitive mechanism, HAEC were pretreated withthe antioxidant pyrrolidine dithiocarbamate (PDTC, 50 μM) for 30 minutesand then the cells were independently incubated with linoleic acid,arachidonic acid, 13-HPODE, and 15-HPETE (all 7.5 μM) for 8 hours. Itwas determined that PDTC suppressed the gene expression of VCAM-1induced by the polyunsaturated fatty acids and their oxidizedcounterparts. This indicates that the induction is mediated by aoxidized signal molecule, and that the induction is prevented when theoxidation of the molecule is blocked (i.e., the oxidation does notoccur), reversed (i.e., the signal molecule is reduced), or itsinteraction with a target protein prevented, perhaps through a redoxcomplex.

To determine whether the selective induction of VCAM-1 by PUFAs andtheir oxidized metabolites is observed at the mRNA level, HAEC wereincubated with linoleic acid or 13-HPODE. Linoleic acid and 13-HPODEinduced VCAM-1 mRNA accumulation that was similar to levels induced byTNF-α. In contrast, there was no induction of ICAM-1 or E-selectin geneexpression at the MRNA level in HAEC incubated with linoleic acid or13-HPODE. The findings mimic those found at the cell-surface level.These results indicate that pretranslational regulatory mechanismsmediate induction of VCAM-1 gene expression by polyunsaturated fattyacids and their oxidative metabolites.

It was also desired to determine whether polyunsaturated fatty acidswork as a primary signal or operate through a regulatory proteininvolving the cytokine IL-4 in inducing VCAM-1 gene expression. Toinvestigate whether newly synthesized proteins such as IL-4 are involvedin the synthesis and gene expression of VCAM-1 induced by PUFAs such aslinoleic acid, HAEC were incubated with 13-HPODE (7.5 μM) and exposed tothe protein synthesis inhibitor, cycloheximide. There was no inhibitionof mRNA accumulation of VCAM-1 by cycloheximide in HAEC incubated with13-HPODE. The production of IL-4 by HAEC incubated with linoleic orarachidonic acids and their oxidative metabolites, as determined byELISA was also measured. There was no increase in IL-4 output by HAECincubated with these PUFAs or their oxidized metabolites.

Previous investigations have demonstrated through deletion andheterologous promoter studies that cytokines and non-cytokines activateVCAM-1 gene expression in endothelial cells at least in parttranscriptionally through two NF-kB-like DNA binding elements. It hasalso been demonstrated that PDTC inhibits VCAM-1 gene expression througha redox-sensitive NF-kB like factor. To determine whetherpolyunsaturated fatty acids induce transcriptional activation of thehuman VCAM-1 promoter via a similar mechanism, the chimeric reportergene p288 VCAM-CAT, containing coordinates -288 to +22 of the humanVCAM-1 promoter, was transiently transfected into HAEC. The addition oflinoleic acid (7.5 μM) induced VCAM-1 promoter. The addition of linoleicacid (7.5 μM) induced VCAM-I promoter activity that was over two foldthat of the control and approximately 60% of the maximum signal inducedby TNF-α. Similar results were obtained with the minimalcytokine-inducible promoter of the VCAM-1 gene (p85 VCAM-CAT),containing the -77 and -63 bp NF-kB-like sites. Neither linoleic acidnor TNF-α had any effect on activity using a constitutively expressedpSV₂ CAT construct. PDTC inhibited the transcriptional activation ofboth VCAM-1 promoter constructs induced by linoleic acid. The dataindicate that analogous to TNF-α, polyunsaturated fatty acids such aslinoleic acid induce the transcriptional activation of VCAM-1 through anNF-kB-like redox-sensitive mechanism.

To determine whether polyunsaturated fatty acids and their oxidativemetabolites regulate VCAM-1 promoter activity through an NF-kB-liketranscriptional regulatory factor, nuclear extracts from HAEC wereassayed for DNA binding activity to a double-stranded oligonucleotidecontaining the VCAM-1 NF-kB-like promoter elements located at positions-77 and -63. As shown in FIG. 7, two bands A and C, representingNF-kB-like activity were induced in response to a three hour exposure tolinoleic acid (7.5 μM). Similar findings were observed on exposure tothe cytokine TNF-α (100 U/ml). A weak band B was observed in control(untreated) cells. No induction of NF-kB-like binding was observed withthe monounsaturated fatty acid oleic acid. Pretreatment of the cells forthirty minutes with PDTC inhibited the A and C complex DNA bindingactivity after linoleic acid activation. These findings are similar topreviously reported findings that PDTC blocks the activation of VCAM-1gene expression in HUVEC by inhibiting the activation of these NFKB-like DNA binding proteins.

EXAMPLE 1 Effect of Oxidized and Unoxidized Polyunsaturated Fatty Acidson the Kinetics of the Activation of VCAM-1 Gene Expression

Human aortic endothelial cells (HAEC) were plated in 96 well plates andincubated with linoleic acid (7.5 μM), 13-HPODE (7.5 μM), or TNF-α (100U/ml) at five different time points up to 48 hours. HAEC, obtained fromClonetics (Boston, Mass.), were cultured in Medium 199 supplemented with20% fetal bovine serum (FBS), 16 U/ml heparin, 10 U/ml epidermal growthfactor, 50 μg/ml endothelial cell growth supplement, 2 mM L-glutamine,100 U/ml penicillin, and 100 μg/ml streptomycin. One day before theexperiment, cells were placed in a medium containing 4% FBS. ConfluentHAEC were incubated for up to 48 hours with TNF-α (100 U/ml), orstearic, oleic, linoleic, linolenic, or arachidonic acids (7.5 μM).Similar studies were performed with differing doses of linoleic acid or13-HPODE for an 8 hour period (1-60 μM) (FIG. 2). Quantitation wasperformed by determination of colorimetric conversion at 450 nm of TMB.Studies were performed in triplicate (n=4 for each experimental value).*-value differs (p<0.05) from Control.

As shown in FIG. 1, both linoleic acid and 13-HPODE induced theexpression of VCAM-1. At ten hours after exposure, the amount of cellsurface VCAM-1 induced by linoleic acid and 13-HPODE was greater thanhalf that induced by the cytokine TNF-α.

As shown in FIG. 2, the induction of VCAM-1 by linoleic acid and13-HPODE is concentration sensitive. At a concentration of between 2 and10 μM of these compounds, there is a sharp increase in the amount ofinduced cell surface VCAM-1, which then remains approximately constantup to a concentration of at least 100 μM. It should be observed that thePUFA concentration indicated in FIG. 2 is in addition to that foundendogenously in HAEC.

EXAMPLE 2 Polyunsaturated Fatty Acids Induce Gene Expression of VCAM-1but not ICAM-1 or E-selectin.

The cell surface expression of VCAM-1, ICAM-1, and E-selectin wasmeasured in HAEC by ELISA. HAEC, obtained from Clonetics (California),were cultured in Medium 199 supplemented with 20% fetal bovine serum(FBS), 16 U/ml heparin, 10 U/ml epidermal growth factor, 50 μg/mlendothelial cell growth supplement, 2 mM L-glutamine, 100 U/mlpenicillin, and 100 μg/ml streptomycin. One day before the experiment,cells were placed in a medium containing 4% FBS. Confluent HAEC wereincubated or not for 8 hours with TNF-α (100 U/ml), or stearic, oleic,linoleic, linolenic, or arachidonic acids (7.5 μM). Cell-surfaceexpression of A) VCAM-1, B) ICAM-1, and C) E-selectin was determined byprimary binding with VCAM-1 specific, ICAM-1 specific, and E-selectinspecific mouse antibodies followed by secondary binding with ahorseradish peroxidase-tagged goat anti-mouse (IgG). Quantitation wasperformed by determination of colorimetric conversion at 450 mm of TMB.Studies were performed in triplicate (n=4 for each experimental value).*-value differs (p<0.05) from Control.

As shown in FIG. 3, linoleic acid, linolenic acid, and arachidonic acidsignificantly induced the expression of VCAM-1, but did not induce thecell-surface expression of ICAM-1 or E-selectin. Neither stearic acidnor oleic acid induced the expression of VCAM-1, ICAM-1, or E-selectin.TNF-α strongly induced the expression of all three cell-surfacemolecules.

EXAMPLE 3 The Antioxidant PDTC Suppresses VCAM-1 Induction byPolyunsaturated Fatty Acids and their Oxidative Metabolites.

Confluent HAEC were pretreated in the presence or absence of PDTC(sodium pyrrolidine dithiocarbamate, 50 μM) for thirty minutes. Thecells were then incubated for eight hours with TNF-α (100 U/ml),linoleic or arachidonic acid (7.5 μM), or the fatty acid hydroperoxides13-HPODE (7.5 μM) or 15-HPETE (7.5 μM). The cell surface expression ofVCAM-1 was measured in HAEC by ELISA, as described in Example 1. Studieswere performed in triplicate (n=4 for each experimental value). *-valuediffers (p<0.05) from control.

As indicated in FIG. 4, PDTC suppresses the induction of VCAM-1 bylinoleic acid, 13-HPODE, arachidonic acid and 15-HPETE.

EXAMPLE 4 Acute Induction of VCAM-1 mRNA by Linoleic Acid and 13-HPODE.

HAEC were exposed to linoleic acid (7.5 μM) or 13-HPODE (7.5 μM). TotalRNA was isolated and 20 μg size-fractionated by denaturing 1.0%agarose-formaldehyde gel electrophoresis, transferred to nitrocellulose,and hybridized to either ³² P-labeled human A) VCAM-1 specific or B)β-actin specific cDNA and visualized by autoradiography. After washes,filters were exposed to X-ray film at -70° C. with one intensifyingscreen for 24 hours. Identification of lanes: 1) control; 2) linoleicacid (acute, 8-hour exposure); 3) linoleic (48-hour exposure); 4)13-HPODE (acute, 8-hour exposure); and 5) TNF-α (100 U/ml, 4-hourexposure).

As shown in FIG. 5, both linoleic acid and 13-HPODE induce theproduction of mRNA for VCAM-1 in eight hours. After 48 hours, linoleicacid no longer induces VCAM-1 mRNA.

EXAMPLE 5 Induction of VCAM-1 mRNA by PUFAs is Independent of CellularProtein Synthesis.

HAEC were exposed to either linoleic or arachidonic acid (7.5 μM) in thepresence or absence of cycloheximide (10 μg/ml) for a 4-hour period.Total RNA was isolated and 20 μg was size-fractionated by denaturing1.0% agarose-formaldehyde gel electrophoresis, transferred tonitrocellulose, and hybridized to A) 32P-labeled human VCAM-1 or B)β-actin specific cDNA and then visualized by autoradiography. Afterwashes, filters were exposed to X-ray film at -70° C. with oneintensifying screen for 24 hours.

As indicated in FIG. 6, the induction of VCAM-1 by linoleic andarachidonic acids are independent of cellular protein synthesis.

EXAMPLE 6 Linoleic acid induces transcriptional activation of the VCAM-1promoter by a redox-sensitive NF-kB like factor.

HAEC were split at the ratio to give approximately 60% confluence in100-mm tissue culture plates. HAEC were transfected with either 30 μg ofp288 VCAMCAT, p85 VCAMCAT, or pSV₂ CAT plasmid by the calcium phosphatecoprecipitation technique using standard techniques. After a 24-hourrecovery period, HAEC were pretreated with 50 μM PDTC and after 30minutes exposed to linoleic acid (7.5 μM) or TNF-α (100 U/ml) directlyadded to the plates. After 18 hours, cell extracts were prepared byrapid freeze-thaw in 0.25 M Tris, pH 8.0. Protein of each cell extractwas assayed for chloramphenicol acetyl transferase (CAT) activity (Ac,acetylated; N, nonacetylated chloramphenicol).

FIG. 7 illustrates the results of this experiment. Linoleic acid inducestranscriptional activation of the VCAM-1 promoter by a redox-sensitiveNF-kB like factor. These results are similar to those observed by theactivation of VCAM-1 promotor by cytokines such as TNF-α. This suggeststhat PUFAs act through an oxidized intermediate that also mediates thecytokine activation of VCAM-1.

EXAMPLE 7 Polyunsaturated Fatty Acids Activate NF-kB-like DNA BindingActivities that are Blocked by the Antioxidant PDTC.

Confluent HAEC in media containing 4% FBS (as described in Example 1)were pretreated with PDTC (50 μM) for 30 minutes and then exposed for 3hours to linoleic acid or oleic acid (7.5 μM), or TNF-α (100 U/ml). Fivemicrograms of nuclear extract was incubated with a double-stranded ³²P-labeled wtVCAM, size fractionated on 4% native acrylamide gels, andexposed to autoradiography film at -70° C. for 18 hours. Two bands A andC, representing NF-kB like binding activity are designated. A weak bandB was observed in control (untreated) cells.

FIG. 8 illustrates that linoleic acid induces NF-kB binding activity toVCAM-1 promotor in a redox-sensitive manner. This is analogous tocytokine TNF-α and suggests a similar mechanism of action. TNF-αprobably induces VCAM-1 through a mechanism that is mediated by anox-PUFA.

EXAMPLE 8 Oxidation in a cell-free, media-free setup, by both unoxidizedand oxidized (15-HPETE) arachidonic acid

FIGS. 9A and 9B are bar chart graphs of the relative thiabarbituric acidreactive substances (O.D. 532 nm) of arachidonic acid and 15-HPETE inthe presence or absence of PDTC. The thiobarbituric acid reactivityassay (TBARS) measures the oxidation ability of a material in acell-free, media-free environment. As indicated in the Figures, botharachidonic acid and 15-HPETE showed significant TBARS activity that wasinhibited by PDTC.

III. Method for the Treatment of VCAM-1 Mediated Disorders

The discovery that polyunsaturated fatty acids and their oxidizedmetabolites are selective, redox-sensitive immunomodulators provides abasis for the therapy of disorders that are mediated by VCAM-1 or byredox-sensitive genes.

A method for the treatment of atherosclerosis, post-angioplastyrestenosis, coronary artery diseases, angina, and other cardiovasculardiseases, as well as noncardiovascular inflammatory diseases that aremediated by VCAM-1 is provided that includes the removal, decrease inthe concentration of, or prevention of the formation of oxidizedpolyunsaturated fatty acids, including but not limited to oxidizedlinoleic, linolenic, and arachidonic acids. In an alternativeembodiment, a method for the treatment of these diseases is providedthat includes the prevention of the interaction of a PUFA or ox-PUFAwith a protein or peptide that mediates VCAM-1 expression.

Inhibition of the expression of VCAM-1 can be accomplished in a numberof ways, including through the administration of an antioxidant thatprevent the oxidation of a polyunsaturated fatty acid, by in vivomodification of the metabolism of PUFAs into ox-PUFAs, as described inmore detail below.

1. Administration of Antioxidants

Any compound that reduces an ox-PUFA or which inhibits the oxidation ofPUFA, and which is relatively nontoxic and bioavailable or which can bemodified to render it bioavailable, can be used in this therapy. One ofordinary skill in the art can easily determine whether a compoundreduces an ox-PUFA or inhibits the oxidation of PUFA using standardtechniques.

Dithiocarboxylate Antioxidants

It has been discovered that dithiocarboxylates are useful in thetreatment of atherosclerosis and other cardiovascular and inflammatorydiseases. Dithiocarboxylates, including dithiocarbamates, can be used toblock the ability of cells, including endothelial cells, to expressVCAM-1 or to suppress the expression of a redox-sensitive gene oractivate a gene that is suppressed through a redox-sensitive pathway.

At least one of the compounds, pyrrolidine dithiocarbamate (PDTC),inhibits VCAM-1 gene expression at a concentration of less than 1.0micromolar. This compounds also exhibits preferential toxicity toproliferating or abnormally dividing vascular smooth muscle cells.Another dithiocarbamate, sodiumN-methyl-N-carboxymethyl-N-carbodithioate, also inhibits the expressionof VCAM-1, without significant effect on ICAM-1, but does not exhibitpreferential toxicity to abnormally dividing vascular smooth musclecells. Another dithiocarbamate, sodiumN-methyl-N-carboxymethyl-N-carbodithioate, also inhibits the expressionof VCAM-1, without significant effect on ICAM-1, but does not exhibitpreferential toxicity to abnormally dividing vascular smooth musclecells.

It has been discovered that pyrrolidine dithiocarbamate does notsignificantly block ELAM-1 or ICAM-1 expression, and therefore treatmentwith this compound does not adversely affect aspects of the inflammatoryresponse mediated by ELAM-1 or ICAM-1. Thus, a generalizedimmunosuppression is avoided. This may avoid systemic complications fromgeneralized inhibition of adhesion molecules in the many other celltypes known to express them. Other pharmaceutically acceptable salts ofPDTC are also effective agents for the treatment of cardiovascular andinflammatory disorders.

Dithiocarbamates are transition metal chelators clinically used forheavy metal intoxication. Baselt, R. C., F. W. J. Sunderman, et al.(1977), "Comparisons of antidotal efficacy of sodiumdiethyldithiocarbamate, D-penicillamine and triethylenetetramine uponacute toxicity of nickel carbonyl in rats." Res Commun Chem PatholPharmacol 18(4): 677-88; Menne, T. and K. Kaaber (1978), "Treatment ofpompholyx due to nickel allergy with chelating agents." ContactDermatitis 4(5): 289-90; Sunderman, F. W. (1978), "Clinical response totherapeutic agents in poisoning from mercury vapor" Ann Clin Lab Sci8(4): 259-69; Sunderman, F. W. (1979), "Efficacy of sodiumdiethyldithiocarbamate (dithiocarb) in acute nickel carbonyl poisoning."Ann Clin Lab Sci 9(1): 1-10; Gale, G. R., A. B. Smith, et al. (1981),"Diethyldithiocarbamate in treatment of acute cadmium poisoning." AnnClin Lab Sci 11(6): 476-83; Jones, M. M. and M. G. Cherian (1990), "Thesearch for chelate antagonists for chronic cadmium intoxication."Toxicology 62(1): 1-25; Jones, S. G., M. A. Basinger, et al. (1982), "Acomparison of diethyldithiocarbamate and EDTA as antidotes for acutecadmium intoxication." Res Commun Chem Pathol Pharmacol 38(2): 271-8;Pages, A., J. S. Casas, et al. (1985), "Dithiocarbamates in heavy metalpoisoning: complexes of N,N-di(1-hydroxyethyl)dithiocarbamate withZn(II), Cd(II), Hg(II), CH3Hg(II), and C6H5Hg(II).: J. Inorg Biochem25(1): 35-42; Tandon, S. K., N. S. Hashmi, et al. (1990), "Thelead-chelating effects of substituted dithiocarbamates." Biomed EnvironSci 3(3): 299-305.

Dithiocarbamates have also been used adjunctively in cis-platinumchemotherapy to prevent renal toxicity. Hacker, M. P., W. B. Ershler, etal. (1982). "Effect of disulfiram (tetraethylthiuram disulfide) anddiethyldithiocarbamate on the bladder toxicity and antitumor activity ofcyclophosphamide in mice." Cancer Res 42(11): 4490-4. Bodenner, 1986#733; Saran, M. and Bors, W. (1990). "Radical reactions in vivo--anoverview." Radiat. Environ. Biophys. 29(4):249-62.

A dithiocarbamate currently used in the treatment of alcohol abuse isdisulfiram, a dimer of diethyldithiocarbamate. Disulfuram inhibitshepatic aldehyde dehydrogenase. Inoue, K., and Fukunaga, et al., (1982)."Effect of disulfiram and its reduced metabolite, diethyldithiocarbamateon aldehyde dehydrogenase of human erythrocytes." Life Sci 30(5):419-24.

It has been reported that dithocarbamates inhibit HIV virus replication,and also enhance the maturation of specific T cell subpopulations. Thishas led to clinical trials of diethyldithiocarbamate in AIDs patientpopulations. Reisinger, E., et al., (1990). "Inhibition of HIVprogression by dithiocarb." Lancet 335: 679.

Dithiocarboxylates are compounds of the structure A-SC(S)-B, which aremembers of the general class of compounds known as thiol antioxidants,and are alternatively referred to as carbodithiols or carbodithiolates.It appears that the --SC(S)-- moiety is essential for therapeuticactivity, and that A and B can be any group that does not adverselyaffect the efficacy or toxicity of the compound.

In an alternative embodiment, one or both of the sulfur atoms in thedithiocarbamate is replaced with a selenium atom. The substitution ofsulfur for selenium may decrease the toxicity of the molecule in certaincases, and may thus be better tolerated by the patient.

A and B can be selected by one of ordinary skill in the art to impartdesired characteristics to the compound, including size, charge,toxicity, and degree of stability, (including stability in an acidicenvironment such as the stomach, or basic environment such as theintestinal tract). The selection of A and B will also have an importanteffect on the tissue-distribution and pharmacokinetics of the compound.In general, for treatment of cardiovascular disease, it is desirablethat the compound accumulate, or localize, in the arterial intimal layercontaining the vascular endothelial cells. The compounds are preferablyeliminated by renal excretion.

An advantage in administering a dithiocarboxylate pharmaceutically isthat it does not appear to be cleaved enzymatically in vivo bythioesterases, and thus may exhibit a prolonged halflife in vivo.

In a preferred embodiment, A is hydrogen or a pharmaceuticallyacceptable cation, including but not limited to sodium, potassium,calcium, magnesium, aluminum, zinc, bismuth, barium, copper, cobalt,nickel, or cadmium; a salt-forming organic acid, typically a carboxylicacid, including but not limited to acetic acid, oxalic acid, tartaricacid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannicacid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, naphthalenedisulfonic acid, or polygalacturonic acid; or a cationformed from ammonia or other nitrogenous base, including but not limitedto a nitrogenous heterocycle, or a moiety of the formula NR⁴ R⁵ R⁶ R⁷,wherein R⁴, R⁵, R⁶, and R⁷ are independently hydrogen, C₁₋₆ linear,branched, or (in the case of C₄₋₆) cyclic alkyl, hydroxy(C₁₋₆)alkyl(wherein one or more hydroxyl groups are located on any of the carbonatoms), or aryl, N,N-dibenzylethylene-diamine, D-glucosamine, choline,tetraethylammonium, or ethylenediamine.

In another embodiment, A can be a physiologically cleavable leavinggroup that can be cleaved in vivo from the molecule to which it isattached, and includes but is not limited acyl (including acetyl,propionyl, and butyryl), alkyl, phosphate, sulfate or sulfonate.

In one embodiment, B is alkyl, alkenyl, alkynyl, alkaryl, aralkyl,haloalkyl, haloalkenyl, haloalkynyl, aryl, alkaryl, hydrogen, C₁₋₆alkoxy-C₁₋₁₀ alkyl, C₁₋₆ alkylthio-C₁₋₁₀ alkyl, NR² R³, --(CHOH)_(n) CH₂OH, wherein n is 0, 1, 2, 3, 4, 5, or 6, --(CH₂)_(n) CO₂ R¹, includingalkylacetyl, alkylpropionyl, and alkylbutyryl, or hydroxy(C₁₋₆)alkyl-(wherein one or more hydroxyl groups are located on any of the carbonatoms).

In another embodiment, B is NR² R³, wherein R² and R³ are independentlyalkyl; --(CHOH)_(n) (CH₂)_(n) OH, wherein n is 0, 1, 2, 3, 4, 5, or 6;--(CH₂)_(n) CO₂ R¹, --(CH₂)_(n) CO₂ R⁴ ; hydroxy(C₁₋₆)alkyl-; alkenyl(including but not limited to vinyl, allyl, and CH₃ CH═CH--CH₂₋ CH₂);alkyl(CO₂ H), alkenyl(CO₂ H), alkynyl(CO₂ H), or aryl, wherein the arylgroup can be substituted as described above, notably, for example, witha NO₂, CH₃, t-butyl, CO₂ H, halo, or p-OH group; or R² and R³ cantogether constitute a bridge such as --(CH₂)m--, wherein m is 3, 4, 5,or 6, and wherein R⁴ is alkyl, aryl, alkaryl, or aralkyl, includingacetyl, propionyl, and butyryl.

In yet another embodiment, B can be a heterocyclic or alkylheterocyclicgroup. The heterocycle can be optionally partially or totallyhydrogenated. Nonlimiting examples are those listed above, includingphenazine, phenothiazine, pyridine and dihydropyridine.

In still another embodiment, B is the residue of apharmaceutically-active compound or drug. The term drug, as used herein,refers to any substance used internally or externally as a medicine forthe treatment, cure, or prevention of a disease or disorder. Nonlimitingexamples are drugs for the treatment or prevention of cardiovasculardisease, including antioxidants such as probucol; nicotinic acid; agentsthat prevent platelets from sticking, such as aspirin; antithromboticagents such as coumadin; calcium channel blockers such as varapamil,diltiazem, and nifedipine; angiotensin converting enzyme (ACE)inhibitors such as captopril and enalopril, β-blockers such aspropanalol, terbutalol, and labetalol, nonsteroidal antiinflammatoriessuch as ibuprofen, indomethacin, fenoprofen, mefenamic acid, flufenamicacid, sulindac, or corticosteriods. The --C(S)SA group can be directlyattached to the drug, or attached through any suitable linking moiety.

In another embodiment, the dithiocarbamate is an amino acid derivativeof the structure AO₂ C--R⁹ --NR¹⁰ --C(S)SA, wherein R₉ is a divalent Bmoiety, a linking moiety, or the internal residue of any of thenaturally occurring amino acids (for example, CH₃ CH for alanine, CH₂for glycine, CH(CH₂)₄ NH₂ for lysine, etc.), and R¹⁰ is hydrogen orlower alkyl.

B can also be a polymer to which one or more dithiocarbamate groups areattached, either directly, or through any suitable linking moiety. Thedithiocarbamate is preferably released from the polymer under in vivoconditions over a suitable time period to provide a therapeutic benefit.In a preferred embodiment, the polymer itself is also degradable invivo. The term biodegradable or bioerodible, as used herein, refers to apolymer that dissolves or degrades within a period that is acceptable inthe desired application (usually in vivo therapy), usually less thanfive years, and preferably less than one year, on exposure to aphysiological solution of pH 6-8 having a temperature of between 25° and37° C. In a preferred embodiment, the polymer degrades in a period ofbetween 1 hour and several weeks, according to the application.

A number of degradable polymers are known. Nonlimiting examples arepeptides, proteins, nucleoproteins, lipoproteins, glycoproteins,synthetic and natural polypeptides and polyamino acids, including butnot limited to polymers and copolymers of lysine, arginine, asparagine,aspartic acid, cysteine, cystine, glutamic acid, glutamine,hydroxylysine, serine, threonine, and tyrosine; polyorthoesters,including poly(α-hydroxy acids), for example, polylactic acid,polyglycolic acid, poly(lactide-co-glycolide), polyanhydrides, albuminor collagen, a polysaccharide containing sugar units such as lactose,and polycaprolactone. The polymer can be a random or block copolymer.

B can also be a group that enhances the water solubility of thedithiocarbamate, for example, -lower alkyl--O--R⁸, wherein R⁸ is --PO₂(OH)⁻ M⁺ or PO₃ (M⁺)₂ wherein M⁺ is a pharmaceutically acceptablecation; --C(O)(CH₂)₂ CO₂ ⁻ M⁺, or --SO₃ ⁻ M⁺ ; -loweralkylcarbonyl-lower alkyl; -carboxy lower alkyl; -lower alkylamino-loweralkyl; N,N-di-substituted amino lower alkyl-, wherein the substituentseach independently represent lower alkyl; pyridyl-lower alkyl-;imidazolyl-lower alkyl-; imidazolyl-Y-lower alkyl wherein Y is thio oramino; morpholinyl-lower alkyl; pyrrolidinyl-lower alkyl;thiazolinyl-lower alkyl-; piperidinyl-lower alkyl; morpholinyl-lowerhydroxyalkyl; N-pyrryl; piperazinyl-lower alkyl; N-substitutedpiperazinyl-lower alkyl, wherein the substituent is lower alkyl;triazolyl-lower alkyl; tetrazolyl-lower alkyl; tetrazolylamino-loweralkyl; or thiazolyl-lower alkyl.

In an alternative embodiment, a dimer such as B--C(S)S--SC(S)--B can beadministered.

Nonlimiting examples of dithiocarbamates are those of the structure:##STR2## Dihydropyridine, calcium channel blocker nefidipine, andderivatives, substituted and unsubstituted.

Dithiocarboxylates should be chosen for use in treating atherosclerosisand other cardiovascular and inflammatory diseases that have the properlipophilicity to locate at the affected cite. The compound should notcompartmentalize in low turnover regions such as fat deposits. In apreferred embodiment for treatment of cardiovascular disease, thepharmacokinetics of the compound should not be dramatically affected bycongestive heart failure or renal insufficiency.

For topical applications for the treatment of inflammatory skindisorders, the selected compound should be formulated to be absorbed bythe skin in a sufficient amount to render a therapeutic effect to theafflicted site.

The dithiocarboxylate must be physiologically acceptable. In general,compounds with a therapeutic index of at least 2, and preferably atleast 5 or 10, are acceptable. The therapeutic index is defined as theEC₅₀ /IC₅₀, wherein EC₅₀ is the concentration of compound that inhibitsthe expression of VCAM-1 by 50% and IC₅₀ is the concentration ofcompound that is toxic to 50% of the target cells. Cellular toxicity canbe measured by direct cell counts, trypan blue exclusion, or variousmetabolic activity studies such as 3H-thymidine incorporation, as knownto those skilled in the art. The therapeutic index of PDTC in tissueculture is over 100 as measured by cell toxicity divided by ability toinhibit VCAM-1 expression activated by TNFa, in HUVE cells. Initialstudies on the rapidly dividing cell type HT-18 human glioma demonstrateno toxicity at concentrations 100-fold greater than the therapeuticconcentration. Disulfiram, an orally administered form ofdiethyldithiocarbamate, used in the treatment of alcohol abuse,generally elicits no major clinical toxicities when administeredappropriately.

There are a few dithiocarbamates that are known to be genotoxic. Thesecompounds do not fall within the scope of the present invention, whichis limited to the administration of physiologically acceptablematerials. An example of a genotoxic dithiocarbamate is the fungicidezinc dimethyldithiocarbamate. Further, the anticholinesterase propertiesof certain dithiocarbamates can lead to neurotoxic effects. Miller, D.(1982). Neurotoxicity of the pesticidal carbamates. Neurobehav. Toxicol.Teratol. 4(6): 779-87.

The term dithiocarboxylate as used herein specifically includes, but isnot limited to, dithiocarbamates of the formulas:

    R.sup.1 SC(S)NR.sup.2 R.sup.3 or R.sup.2 R.sup.3 N(S)CS--SC(S)NR.sup.2 R.sup.3

wherein R¹ is H or a pharmaceutically acceptable cation, including butnot limited to sodium, potassium, or NR⁴ R⁵ R⁶ R⁷, wherein R⁴, R⁵, R⁶,and R⁷ are independently hydrogen, C₁₋₆ linear, branched, or cyclicalkyl, hydroxy(C₁₋₆)alkyl (wherein one or more hydroxyl groups arelocated on any of the carbon atoms), or aryl, and

R² and R³ are independently C₁₋₁₀ linear, branched or cyclic alkyl;--(CHOH)_(n) (CH₂)_(n) OH, wherein n is 0, 1, 2, 3, 4, 5, or 6;--(CH₂)_(n) CO₂ R¹, --(CH₂)_(n) CO₂ R⁴ ; hydroxy(C₁₋₆)alkyl-, or R² andR³ together constitute a bridge such as --(CH₂)m--, wherein m is 3-6,and wherein R⁴ is alkyl, aryl, alkaryl, or aralkyl, including acetyl,propionyl, and butyryl.

Specific examples of useful dithiocarbamates, illustrated in FIG. 15,include sodium pyrrolidine-N-carbodithioate, sodiumN-methyl-N-carboxymethyl-N-carbodithioate, trisodiumN,N-di(carboxymethyl)-N-carbodithioate, sodiumN-methyl-D-glucamine-N-carbodithioate, sodiumN,N-diethyl-N-carbodithioate (sodium diethyldithiocarbamate), and sodiumN,N-diisopropyl-N-carbodithioate.

The active dithiocarboxylates and in particular, dithiocarbamates areeither commercially available or can be prepared using known methods.

II. Biological Activity

The ability of dithiocarboxylates to inhibit the expression of VCAM-1can be measured in a variety of ways, including by the methods set outin detail below in Examples 9 to 15. For convenience, Examples 9-11 and14-15 describe the evaluation of the biological activity of sodiumpyrrolidine-N-carbodithioate (also referred to as PDTC). These examplesare not intended to limit the scope of the invention, which specificallyincludes the use of any of the above-described compounds to treatatherosclerosis, and other types of inflammation and cardiovasculardisease mediated by VCAM-1. Any of the compounds described above can beeasily substituted for PDTC and evaluated in similar fashion.

Examples 12 and 13 provide comparative data on the ability of a numberof dithiocarbamates to inhibit the gene expression of VCAM-1. Theexamples below establish that the claimed dithiocarbamates specificallyblock the ability of VCAM-1 to be expressed by vascular endothelialcells in response to many signals known to be active in atherosclerosisand the inflammatory response.

Experimental Procedures

Cell Cultures HUVE cells were isolated from human umbilical veins thatwere cannulated, perfused with Hanks solution to remove blood, and thenincubated with 1% collagenase for 15 minutes at 37° C. After removal ofcollagenase, cells were cultured in M199 medium supplemented with 20%fetal bovine serum (HyClone), 16 μg/ml heparin (ESI Pharmaceuticals,Cherry Hill, N.J.), 50 μg/ml endothelial cell growth supplement(Collaborative Research Incorporated, Bedford Mass.), 25 mM HepesBuffer, 2 mM L-glutamin, 100 μg/ml penicillin and 100 μg/ml streptomycinand grown at 37° C. on tissue culture plates coated 0.1% gelatin. Cellswere passaged at confluency by splitting 1:4. Cells were used within thefirst 8 passages.

Incubation with Cytokines and Other Reagents Confluent HUVE cells werewashed with phosphate buffered saline and then received fresh media. Theindicated concentrations of PDTC were added as pretreatment 30 minutesbefore adding cytokines. Cytokines and other inducers were directlyadded to medium for the times and at the concentrations indicated ineach experiment. Human recombinant IL-lb was the generous gift of UpjohnCompany (Kalamazoo, Mich.). TNFa was obtained from Bohringer Engelheim.Bacterial lipopolysaccharide (LPS), polyinosinic acid: polycitidilicacid (Poly I:C), and pyrrolidine dithiocarbamate (PDTC) were obtainedfrom Sigma Chemical (St. Louis, Mo.). All other reagents were of reagentgrade.

RNA Isolation: Total cellular RNA was isolated by a single extractionusing an acid guanidium thiocyanate-phenol-chloroform mixture. Cellswere rinsed with phosphate buffered saline and then lysed with 2 ml ofguanidium isothiocyanate. The solution was acidified with 0.2 ml ofsodium acetate (pH 4.0) and then extracted with 2 ml phenol and 0.4 mlchloroform:isoamyl alcohol (24:1). The RNA underwent two ethanolprecipitations prior to being used for Northern blot analysis.

Northern Blot Analysis: Total cellular RNA (20 μg) was size fractionatedusing 1% agarose formaldehyde gels in the presence of 1 ug/ml ethidiumbromide. The RNA was transferred to a nitrocellulose filter andcovalently linked by ultraviolet irradiation using a Stratlinker UVcrosslinker (Stratagene, La Jolla, Calif.). Hybridizations wereperformed at 42° C. for 18 hours in 5× SSC (1×=150 mM NaCl, 15 mM Nacitrate), 1% sodium dodecyl sulfate, 5× Denhardt solution, 50%formamide, 10% dextran sulfate and 100 ug/ml of sheared denatured salmonsperm DNA. Approximately 1-2×10⁶ cpm/ml of labeled probe (specificactivity>108 cpm/ug DNA) were used per hybridization. Followinghybridization, filters were washed with a final stringency of 0.2× SSCat 55° C. The nitrocellulose was stripped using boiled water prior torehybridization with other probes. Autoradiography was performed with anintensifying screen at -70° C.

³² Probes: ³² P labeled DNA probes were made using the random primeroligonucleotide method. The ICAM-1 probe was an Eco RI fragment of humancDNA. The ELAM-1 probe was a 1.85 kb Hind III fragment of human cDNA.The VCAM-1 probe was a Hind III-Xho I fragment of the human cDNAconsisting of nucleotide 132 to 1814.

Enzyme Linked Immunosorbent Assay (ELISA): HUVE cells were plated on96-well tissue culture plates 48 to 72 hours before the assay. Primaryantibodies in M199 with 5% FBS were added to each well and incubated onehour at 37° C. The cells were then washed and incubated for one hourwith peroxidase conjugated goat anti-mouse IgG (Bio Rad) diluted 1/500in M199 with 5% FBS. The wells were then washed and binding of antibodywas detected by the addition of 100 μl of 10 mg/ml3,3,5,5'-tetramethyl-benzidine (Sigma) with 0.003% H₂ O₂. The reactionwas stopped by the addition of 25 μl of 8N sulfuric acid. Plates wereread on an ELISA reader (Bio Rad) at OD 450 nm after blanking on rowsstained only with second step antibody. Data represent the means oftriplicate.

Antibodies: Monoclonal antibody (MAb) 4B9 recognizing vascular celladhesion molecule-1 (VCAM-1) was the generous gift of Dr. John Harlan(University of Washington). MAb E9AlFl recognizing endothelial celladhesion molecule (ELAM-1) was the generous gift of Dr. Swerlick (EmoryUniversity). Hybridomas producing mAb 84H10 recognizing intercellularadhesion molecule 1 (ICAM-1) are routinely grown in our laboratory andantibody was used as tissue culture supernatant.

EXAMPLE 9 PDTC Blocks IL-lb Mediated Induction of HUVEC VCAM-1, but notICAM-1 or ELAM-1, mRNA Accumulation

To determine whether the oxidative state of the endothelial cell canalter the basal or induced expression of cell adhesion molecule genes,cultured human vascular endothelial cells were exposed to the inducingcytokine, IL-lb (10 U/ml) in the presence or absence of the thiolatedmetal chelating antioxidant, pyrrolidine dithiocarbamate (PDTC, 50 μM)for up to 24 hours. As shown in FIG. 10, IL-lb alone (lanes 2, 4, 6, 8)induces the expected rapid and transient induction of VCAM-1 (Panel A),E-selectin (ELAM-1, Panel B) and ICAM-1 (Panel C) mRNA accumulation, allof which peak at four hours. However, in the presence of PDTC, IL-lbinduction of VCAM-1 mRNA accumulation is dramatically inhibited by over90% (panel A, lanes 3, 5, 7, 9). In contrast, although IL-lb mediatedinduction of ELAM-1 is slightly inhibited at 2 and 24 hours (comparelane 2 and 3, 8 and 9, panel B), PDTC does not inhibit the induction at4 and 8 hours (lane 5 and 7, panel B). IL-lb mediated induction ofICAM-1 mRNA accumulation is not affected (panel B, lanes 3, 5, 7, 9).Indeed, a mild augmentation of IL-lb induction of ICAM-1 mRNAaccumulation (˜30%) is observed (compare lanes 4 and 5, panel B).Equivalent amounts of nitrocellulose transferred RNA per lane wasconfirmed by ethidium bromide staining and visualization.

A dose-response analysis was performed to determine whether PDTCinhibits the induction of VCAM-1 gene expression by IL-lb in a dosedependent manner. As shown in FIG. 11, PDTC inhibits IL-lb mediatedinduction of VCAM-1 gene expression with a steep dose-response curve(FIG. 11, panel A) with a calculated EC₅₀ of approximately 10 μM, whilePDTC does not inhibit IL-lb mediated induction of ELAM-1 expression withthese concentrations (FIG. 11, panel B). The IL-lb mediated induction ofICAM-1 mRNA accumulation is enhanced by PDTC with the concentrationhigher than 0.5 μM (FIG. 2, compare lane 2 and lane 4-7, panel C).

These data demonstrate that IL-lb utilizes a dithiocarboxylate, and inparticular, a dithiocarbamate sensitive step as part of its signalingmechanism in the induction of VCAM-1 gene expression. The data alsoappear to indicate that this dithiocarbamate sensitive step does notplay a significant role in the IL-lb mediated induction of ELAM-1 orICAM-1 gene expression.

EXAMPLE 10 PDTC Blocks Induction of HUVEC VCAM-1 mRNA Accumulation byMultiple Stimuli

To determine whether other well-described activators of VCAM-1 geneexpression also utilize a PDTC sensitive step, three distinct classes ofactivators were tested: another classic receptor mediated inducing agent(TNFa), a non-receptor mediated inducer (lipopolysaccharide (LPS)) and arecently described novel inducer (double stranded RNA, poly(I:C)). Inall three cases, PDTC dramatically inhibited the induction of VCAM-1mRNA accumulation in HUVECs after four hours (FIG. 12, Panel A).Although the TNFa mediated ELAM-1 gene expression is suppressed to someextent (FIG. 12 lane 1 and 2, panel B), LPS and poly(I:C) mediatedELAM-1 mRNA accumulation was unaffected (FIG. 12 lane 3-6, panel B). Theinduction of ICAM-1 mRNA accumulation was unaffected (FIG. 12, Panel C).This data indicates that structurally distinct inducing agents, actingthrough distinct pathways, share a common regulatory step specific forthe induction of VCAM-1 gene expression.

EXAMPLE 11 PDTC Blocks HUVE Cell Surface Expression of VCAM-1 Induced byMultiple Stimuli

To determine whether, like its mRNA, the induction of endothelial cellsurface protein expression of VCAM-1 could also be inhibited by PDTC,monoclonal antibodies were used in an ELISA assay to quantitate theinduction of cell surface VCAM-1 and ICAM-1 in cultured HUVE cells. Asshown in FIG. 13, multiple classes of activating agents, in the absenceof PDTC (-PDTC), induce the rapid and transient accumulation of VCAM-1(top left panel) at the cell surface peaking at six hours. In thepresence of PDTC (+PDTC, top right panel), the induction of cell surfaceexpression of VCAM-1 by all agents tested is dramatically inhibited(80-90%). In contrast, the induced expression of cell surface ICAM-1 isunaffected under identical conditions (bottom left and right panels).

These data demonstrate that, like its mRNA accumulation, cell surfaceVCAM-1 expression are selectively inhibited by dithiocarbamates and thatmultiple classes of activating agents utilize a similar, dithiocarbamatesensitive mechanism to induce VCAM-1 gene expression.

EXAMPLE 12 Comparative Effectiveness of Antioxidants in Blocking TNFaInduction of VCAM-1

To determine whether structurally similar or dissimilar antioxidantscould also inhibit VCAM-1 gene expression, and with what potency, HUVEcells were exposed to TNFa for six hours in the presence or absence ofdifferent concentrations of four different antioxidants. As shown inFIG. 14, both diethyldithiocarbamate (DETC) and N-acetyl cysteine (NAC)inhibited VCAM-1 expression at concentrations of 5 μM and 30 μM,respectively. In contrast, PDTC (PDTC) was effective between 5 and 50μM. The iron metal chelator, desferroximine, had no effect at theconcentrations tested.

EXAMPLE 13 PDTC Inhibits TNF Induction of VCAM-1/VLA-4 Mediated Adhesion

The ability of a variety of antioxidants to inhibit TNF-α induction ofVCAM-1 in HUVE cells was evaluated by the method set out in Example 12.FIG. 15 is a graph of the relative VCAM-1 cell surface expression (O.D.595 nM) in TNF-α activated HUVE cells versus concentrations of PTDC(sodium N-pyrrolidine dithiocarbamate), DIDTC (sodiumN,N-diethyl-N-carbodithioate), SarDTC (sodiumN-methyl-N-carboxymethyl-N-carbodithioate), IDADTC (trisodiumN,N-di(carboxymethyl)-N-carbodithioate), MGDTC (sodiumN-methyl-D-glucamine-N-carbodithioate), MeOBGDTC (sodiumN-(4-methoxybenzyl)-D-glucamine-N-carbodithioate), DEDTC (sodiumN,N-diethyl-N-carbodithioate), Di-PDTC (sodiumN,N-diisopropyl-N-carbodithioate), and NAC is (N-acetyl cysteine).

EXAMPLE 13 PDTC Inhibits TNF Induction of VCAM-1/VLA-4 Mediated Adhesion

In order to define whether PDTC inhibition of VCAM-1 regulation isassociated with functional consequences, the binding of Molt-4 cells toHUVEC cells either unstimulated or stimulated with TNFa (1000 U/ml) wasexamined for six hours in the presence or absence of PDTC. Molt-4 cellshave been previously shown to bind to activated HUVEC via a VCAM-1dependent mechanism. As shown in FIG. 16, the percentage of Molt-4binding to HUVEC cells decreased when PDTC was present in the media.

EXAMPLE 14 PDTC Inhibits Monocyte Binding to the Thoracic Aorta ofCholesterol Fed Rabbits

An experiment was performed to determine whether the thiol antioxidantPDTC would be efficacious in blocking the first monocyte bindingcomponent of atherosclerosis in an experimental animal model. One matureNew Zealand white rabbit (3.5 Kg) received an intravenous injection ofPDTC (20 mg/Kg, as a concentration of 20 mg/ml in PBS) once daily for 5days. Injections were given via an indwelling cannula in the marginalear vein, which as kept patent by flushing with heparinized salinesolution. The PDTC solution was mixed fresh daily or on alternate days(stored light-protected at 4° C.), and filtered (0.45 mm pore filter)just prior to use. After the first injection, when the cannula wasplaced, the drug was administered with the rabbit in the conscious statewithout apparent discomfort or other ill effect. On the second day ofinjections, the rabbit was given chow containing 1% cholesterol byweight, which was continued throughout the remainder of the experiment.On the fifth day, the animal was euthanized and the thoracic aorta wasexcised and fixed. After appropriate preparation, the sample was imagedon the lower stage of an ISI DS-130 scanning electron microscopeequipped with a LaB emitter. Using dual-screen imaging and a transparentgrid on the CRT screen, 64 adjacent fields at a 620× magnification wereassessed, to cover an area of ˜1.3 mm². Within each field, the number ofadherent leukocytes (WBC) and erythrocytes (RBC) were counted andrecorded.

The data from the arch sample are as follows: 5 WBC and ˜25 RBC per 1.3mm² field. This level of WBC adhesion is similar to control animals fedregular chow (about 7 per field have been seen in arch and thoracicsamples from 2 `negative control` experiments). `Positive control`rabbits fed 1% cholesterol for 4 days but not given antioxidant showabout a 5-fold increase in adhesion, to 38 WBC/1.3 mm². A considerableamount of mostly cell-sized debris was observed adherent to each archsample. It is unclear whether this material is an artifact ofpreparation, or was present in vivo, and if so, whether it is related toPDTC administration. These studies suggest that PDTC infusions caneffectively block initial monocyte adhesion to the aortic endothelium.

EXAMPLE 15 Inhibition of BSA 13-HPODE Adducts with PDTC

FIG. 18 is a bar chart graph of the effect of PTDC on the formation offluorescent adducts of BSA and 13-HPODE, as measured in fluorescentunits versus micromolar concentration of PDTC. One micromolar of13-HPODE was incubated with 200 micrograms of BSA in the presence ofPDTC for six days. Fluorescence was measured at 430-460 nm withexcitation at 330-360 nm. For details of the assay, see Freebis, J.,Parthasarathy, S., Steinberg, D, Proceedings of the National Academy ofSciences 89, 10588-10592, 1992. In a typical reaction 100 nmols of LOOH(generated by the lipoxygenase catalyzed oxidation of linoleic acid) inincubated with 100 μg of bovine serum albumin for 48 to 72 hours and theformation of fluorescent products are followed by measuring thefluorescent spectrum with excitation at 360 nm and emission between 390and 500 nm.

As indicated, PDTC decreases the concentration of fluorescent adducts ofBSA and 13-HPODE.

FIG. 19 is a graph of the effect of PTDC on the formation of fluorescentadducts of BSA and ox-PUFA as a function of wavelength (nm) andconcentration of PDTC. As the concentration of PDTC increases, thequantity of fluorescent adducts decrease.

EXAMPLE 16 Effect of PDTC on the oxidation of LDL by horseradishperoxidase

FIG. 20 is a graph of the effect of PDTC on the oxidation of LDL byhorseradish peroxidase (HRP), as measured over time (minutes) forvarying concentrations of PDTC. The oxidation of LDL was followed bymeasuring the oxidation of the fatty acid components of LDL asdetermined by the increase in optical density at 234 nm. When apolyunsaturated fatty acid is oxidized, there is a shift of double bondsresulting in the formation of conjugated dienes which absorb at 234 nm.The intercept of the initiation and propagation curve (lag phase) issuggested to be a measure of the oxidizability of LDL. Higher the lagphase, more resistant is the LDL to oxidation. Typically 100 μg of humanLDL is incubated with 5 μM H₂ O₂ and the increase in absorption of 234nm is followed.

It is observed that after an incubation period, PDTC inhibits theoxidation of LDL by HRP in a manner that is concentration dependent.

EXAMPLE 17 Effect of PDTC on the cytokine-induced formation of ox-PUFA

FIG. 21 is a chart of the effect of PDTC on the cytokine-inducedformation of ox-PUFA in human aortic endothelial cells. As indicated,both TNF-α and IL-lB causes the oxidation of linoleic acid toox-linoleic acid. The oxidation is significantly prevented by PDTC.

2. Modification of the Synthesis and Metabolism of PUFAs and ox-PUFAs

Inhibition of the expression of VCAM-1 can be accomplished via amodification of the metabolism of PUFAs into ox-PUFAs. For example, anumber of enzymes are known to oxidize unsaturated materials, includingperoxidases, lipoxygenases, cyclooxygenases, and cytochrome P450. Theinhibition of these enzymes may prevent the oxidation of PUFAs in vivo.PUFAs can also be oxidized by metal-dependent nonenzymatic materials.

IV. Method for Modifying the Expression of a Redox-Sensitive Gene

In an alternative embodiment, a method is provided for suppressing theexpression of a redox-sensitive gene or activating a gene that issuppressed through a redox-sensitive pathway, that includesadministering an effective amount of a substance that prevents theoxidation of the oxidized signal, and typically, the oxidation of apolyunsaturated fatty acid. Representative redox-sensitive genes thatare involved in the presentation of an immune response include, but arenot limited to, those expressing cytokines involved in initiating theimmune response (e.g., IL-1β), chemoattractants that promote themigration of inflammatory cells to a point of injury (e.g., MCP-1),growth factors (IL-6, thrombin receptor), and adhesion molecules (e.g.,VCAM-1 and E-selectin).

Given this disclosure, one of ordinary skill in the art will be able toscreen a wide variety of antioxidants for their ability to suppress theexpression of a redox-sensitive gene or activate a gene that issuppressed through a redox-sensitive pathway. All of these embodimentsare intended to fall within the scope of the present invention.

Based on the results of this screening, nucleic acid moleculescontaining the 5' regulatory sequences of the redox-sensitive genes canbe used to regulate or inhibit gene expression in vivo can beidentified. Vectors, including both plasmid and eukaryotic viralvectors, may be used to express a particular recombinant 5' flankingregion-gene construct in cells depending on the preference and judgmentof the skilled practitioner (see, e.g., Sambrook et al., Chapter 16).Furthermore, a number of viral and nonviral vectors are being developedthat enable the introduction of nucleic acid sequences in vivo (see,e.g., Mulligan, 1993 Science, 260, 926-932; U.S. Pat. No. 4,980,286;U.S. Pat. No. 4,868,116; incorporated herein by reference). Recently, adelivery system was developed in which nucleic acid is encapsulated incationic liposomes which can be injected intravenously into a mammal.This system has been used to introduce DNA into the cells of multipletissues of adult mice, including endothelium and bone marrow (see, e.g.,Zhu et al., 1993 Science 261, 209-211; incorporated herein byreference).

The 5' flanking sequences of the redox-sensitive gene can be used toinhibit the expression of the redox-sensitive gene. For example, anantisense RNA of all or a portion of the 5' flanking region of theredox-sensitive gene can be used to inhibit expression of the gene invivo. Expression vectors (e.g., retroviral expression vectors) arealready available in the art which can be used to generate an antisenseRNA of a selected DNA sequence which is expressed in a cell (see, e.g.,U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286). Accordingly, DNAcontaining all or a portion of the sequence of the 5' flanking region ofthe gene can be inserted into an appropriate expression vector so thatupon passage into the cell, the transcription of the inserted DNA yieldsan antisense RNA that is complementary to the mRNA transcript of thegene normally found in the cell. This antisense RNA transcript of theinserted DNA can then base-pair with the normal mRNA transcript found inthe cell and thereby prevent the mRNA from being translated. It is ofcourse necessary to select sequences of the 5' flanking region that aredownstream from the transcriptional start sites for the redox-sensitivegene to ensure that the antisense RNA contains complementary sequencespresent on the mRNA. Antisense RNA can be generated in vitro also, andthen inserted into cells. Oligonucleotides can be synthesized on anautomated synthesizer (e.g., Model 8700 automated synthesizer ofMilligen-Biosearch, Burlington, Mass. or ABI Model 380B). In addition,antisense deoxyoligonucleotides have been shown to be effective ininhibiting gene transcription and viral replication (see e.g., Zamecniket al., 1978 Proc. Natl. Acad. Sci. USA 75, 280-284; Zamecnik et al.,1986 Proc. Natl. Acad. Sci., 83, 4143-4146; Wickstrom et al., 1988 Proc.Natl. Acad. Sci. USA 85, 1028-1032; Crooke, 1993 FASEB J. 7, 533-539.Furthermore, recent work has shown that improved inhibition ofexpression of a gene by antisense oligonucleotides is possible if theantisense oligonucleotides contain modified nucleotides (see, e.g.,Offensperger et. al., 1993 EMBO J. 12, 1257-1262 (in vivo inhibition ofduck hepatitis B viral replication and gene expression by antisensephosphorothioate oligodeoxynucleotides); Rosenberg et al., PCT WO93/01286 (synthesis of sulfurthioate oligonucleotides); Agrawal et al.,1988 Proc. Natl. Acad. Sci. USA 85, 7079-7083 (synthesis of antisenseoligonucleoside phosphoramidates and phosphorothioates to inhibitreplication of human immunodeficiency virus-1); Sarin et al., 1989 Proc.Natl. Acad. Sci. USA 85, 7448-7794 (synthesis of antisensemethylphosphonate oligonucleotides); Shaw et al., 1991 Nucleic Acids Res19, 747-750 (synthesis of 3' exonuclease-resistant oligonucleotidescontaining 3' terminal phosphoroamidate modifications); incorporatedherein by reference).

The sequences of the 5' flanking region of the redox-sensitive gene canalso be used in triple helix (triplex) gene therapy. Oligonucleotidescomplementary to gene promoter sequences on one of the strands of theDNA have been shown to bind promoter and regulatory sequences to formlocal triple nucleic acid helices which block transcription of the gene(see, e.g., 1989 Maher et al., Science 245, 725-730; Orson et al., 1991Nucl. Acids Res. 19, 3435-3441; Postal et al., 1991 Proc. Natl. Acad.Sci. USA 88, 8227-8231; Cooney et al., 1988 Science 241, 456-459; Younget al., 1991 Proc. Natl. Acad. Sci. USA 88, 10023-10026; Duval-Valentinet al., 1992 Proc. Natl. Acad. Sci. USA 89, 504-508; 1992 Blume et al.,Nucl. Acids Res. 20, 1777-1784; 1992 Grigoriev et al., J. Biol. Chem.267, 3389-3395.

Recently, both theoretical calculations and empirical findings have beenreported which provide guidance for the design of oligonucleotides foruse in oligonucleotide-directed triple helix formation to inhibit geneexpression. For example, oligonucleotides should generally be greaterthan 14 nucleotides in length to ensure target sequence specificity(see, e.g., Maher et al., (1989); Grigoriev et al., (1992)). Also, manycells avidly take up oligonucleotides that are less than 50 nucleotidesin length (see e.g., Orson et al., (1991); Holt et al., 1988 Mol. Cell.Biol. 8, 963-973; Wickstrom et al., 1988 Proc. Natl. Acad. Sci. USA 85,1028-1032). To reduce susceptibility to intracellular degradation, forexample by 3' exonucleases, a free amine can be introduced to a 3'terminal hydroxyl group of oligonucleotides without loss of sequencebinding specificity (Orson et al., 1991). Furthermore, more stabletriplexes are formed if any cytosines that may be present in theoligonucleotide are methylated, and also if an intercalating agent, suchas an acridine derivative, is covalently attached to a 5' terminalphosphate (e.g., via a pentamethylene bridge); again without loss ofsequence specificity (Maher et al., (1989); Grigoriev et al., (1992).

Methods to produce or synthesize oligonucleotides are well known in theart. Such methods can range from standard enzymatic digestion followedby nucleotide fragment isolation (see e.g., Sambrook et al., Chapters 5,6) to purely synthetic methods, for example, by the cyanoethylphosphoramidite method using a Milligen or Beckman System 1Plus DNAsynthesizer (see also, Ikuta et al., in Ann. Rev. Biochem. 1984 53,323-356 (phosphotriester and phosphite-triester methods); Narang et al.,in Methods Enzymol., 65, 610-620 (1980) (phosphotriester method).Accordingly, DNA sequences of the 5' flanking region of theredox-sensitive gene described herein can be used to design andconstruct oligonucleotides including a DNA sequence consistingessentially of at least 15 consecutive nucleotides, with or without basemodifications or intercalating agent derivatives, for use in formingtriple helices specifically within the 5' flanking region of aredox-sensitive gene in order to inhibit expression of the gene.

In some cases it may be advantageous to insert enhancers or multiplecopies of the regulatory sequences into an expression system tofacilitate screening of methods and reagents for manipulation ofexpression.

V. Models and Screens

Screens for disorders mediated by VCAM-1 or a redox-sensitive gene arealso provided that include the quantification of surrogate markers ofthe disease. In one embodiment, the level of oxidized polyunsaturatedfatty acid, or other appropriate markers, in the tissue or blood, forexample, of a host is evaluated as a means of assessing the "oxidativeenvironment" of the host and the host's susceptibility to VCAM-1 orredox-sensitive gene mediated disease.

In another embodiment, the level of circulating or cell-surface VCAM-1or other appropriate marker and the effect on that level ofadministration of an appropriate antioxidant is quantified.

In yet another assay, the sensitization of a host's vascular endothelialcells to polyunsaturated fatty acids or their oxidized counterparts isevaluated. This can be accomplished, for example, by challenging a hostwith a PUFA or ox-PUFA and comparing the resulting concentration ofcell-surface or circulating VCAM-1 or other surrogate marker to apopulation norm.

In another embodiment, in vivo models of atherosclerosis or other heartor inflammatory diseases that are mediated by VCAM-1 can be provided byadministering to a host animal an excessive amount of PUFA or oxidizedpolyunsaturated fatty acid to induce disease. These animals can be usedin clinical research to further the understanding of these disorders.

In yet another embodiment of the invention, compounds can be assessedfor their ability to treat disorders mediated by VCAM-1 on the basis oftheir ability to inhibit the oxidation of a polyunsaturated fatty acid,or the interaction of a PUFA or ox-PUFA with a protein target.

This can be accomplished by challenging a host, for example, a human oran animal such as a mouse, to a high level of PUFA or ox-PUFA and thendetermining the therapeutic efficacy of a test compound based on itsability to decrease circulating or cell surface VCAM-1 concentration.Alternatively, an in vitro screen can be used that is based on theability of the test compound to prevent the oxidation of a PUFA, or theinteraction of a PUFA or ox-PUFA with a protein target in the presenceof an oxidizing substance such as a metal, for example, copper, or anenzyme such as a peroxidase, lipoxygenase, cyclooxygenase, or cytochromeP450.

In another embodiment, vascular endothelial cells are exposed to TNF-αor other VCAM-1 inducing material for an appropriate time and thenbroken by any appropriate means, for example by sonication orfreeze-thaw. The cytosolic and membrane compartments are isolated.Radiolabeled PUFA is added to defined amounts of the compartments. Theability of the liquid to convert PUFA to ox-PUFA in the presence orabsence of a test compound is assayed. Intact cells can be used in placeof the broken cell system.

III. Pharmaceutical Compositions

Humans, equine, canine, bovine and other animals, and in particular,mammals, suffering from cardiovascular disorders, and other inflammatoryconditions mediated by VCAM-1 or a redox sensitive gene can be treatedby administering to the patient an effective amount of a compound thatcauses the removal, decrease in the concentration of, or prevention ofthe formation of an oxidized polyunsaturated fatty acids, including butnot limited to oxidized linoleic (C₁₈ Δ⁹,12) , linolenic (C₁₈ Δ⁶,9,12) ,arachidonic (C₂₀ Δ⁵,8,11,14) and eicosatrienoic (C₂₀ Δ⁸,11,14) acids;other oxidation signal; or other active compound, or a pharmaceuticallyacceptable derivative or salt thereof in a pharmaceutically acceptablecarrier or diluent. The active materials can be administered by anyappropriate route, for example, orally, parenterally, intravenously,intradermally, subcutaneously, or topically.

As used herein, the term pharmaceutically acceptable salts or complexesrefers to salts or complexes that retain the desired biological activityof the above-identified compounds and exhibit minimal undesiredtoxicological effects. Nonlimiting examples of such salts are (a) acidaddition salts formed with inorganic acids (for example, hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, andthe like), and salts formed with organic acids such as acetic acid,oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid,benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, naphthalenedisulfonic acid, andpolygalacturonic acid; (b) base addition salts formed with polyvalentmetal cations such as zinc, calcium, bismuth, barium, magnesium,aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and thelike, or with an organic cation formed fromN,N-dibenzylethylene-diamine, D-glucosamine, ammonium,tetraethylammonium, or ethylenediamine; or (c) combinations of (a) and(b); e.g., a zinc tannate salt or the like.

The active compound or a mixture of the compounds are administered inany appropriate manner, including but not limited to orally andintravenously. General range of dosage for any of the above-mentionedconditions will be from 0.5 to 500 mg/kg body weight with a doseschedule ranging from once every other day to several times a day.

The compounds can also be administered directly to the vascular wallusing perfusion balloon catheters following or in lieu of coronary orother arterial angioplasty. As an example, 2-5 mL of a physiologicallyacceptable solution that contains approximately 1 to 500 μM of thecompound or mixture of compounds is administered at 1-5 atmospherespressure. Thereafter, over the course of the next six months during theperiod of maximum risk of restenosis, the active compounds areadministered through other appropriate routes and dose schedules.

Relatively short term treatments with the active compounds are used tocause the "shrinkage" of coronary artery disease lesions that cannot betreated either by angioplasty or surgery. A nonlimiting example of shortterm treatment is two to six months of a dosage ranging from 0.5 to 500mg/kg body weight given at periods ranging from once every other day tothree times daily.

Longer term treatments can be employed to prevent the development ofadvanced lesions in high-risk patients. A long term treatment can extendfor years with dosages ranging from 0.5 to 500 mg/kg body weightadministered at intervals ranging from once every other day to threetimes daily.

The active compounds can also be administered in the period immediatelyprior to and following coronary angioplasty as a means to reduce oreliminate the abnormal proliferative and inflammatory response thatcurrently leads to clinically significant re-stenosis.

The active compounds can be administered in conjunction with othermedications used in the treatment of cardiovascular disease, includinglipid lowering agents such as probucol and nicotinic acid; plateletaggregation inhibitors such as aspirin; antithrombotic agents such ascoumadin; calcium channel blockers such as varapamil, diltiazem, andnifedipine; angiotensin converting enzyme (ACE) inhibitors such ascaptopril and enalopril, and β-blockers such as propanalol, terbutalol,and labetalol. The compounds can also be administered in combinationwith nonsteroidal antiinflammatories such as ibuprofen, indomethacin,fenoprofen, mefenamic acid, flufenamic acid, sulindac. The compound canalso be administered with corticosteriods.

The concentration of active compound in the drug composition will dependon absorption, distribution, inactivation, and excretion rates of thedrug as well as other factors known to those of skill in the art. It isto be noted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed composition. The active ingredient may be administered atonce, or may be divided into a number of smaller doses to beadministered at varying intervals of time.

Oral compositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, dosageunit forms can contain various other materials which modify the physicalform of the dosage unit, for example, coatings of sugar, shellac, orother enteric agents.

The active compound or pharmaceutically acceptable salt or derivativethereof can be administered as a component of an elixir, suspension,syrup, wafer, chewing gum or the like. A syrup may contain, in additionto the active compounds, sucrose as a sweetening agent and certainpreservatives, dyes and colorings and flavors.

The active compound or pharmaceutically acceptable derivatives or saltsthereof can also be administered with other active materials that do notimpair the desired action, or with materials that supplement the desiredaction, such as antibiotics, antifungals, antiinflammatories, orantiviral compounds.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parental preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

Suitable vehicles or carriers for topical application are known, andinclude lotions, suspensions, ointments, creams, gels, tinctures,sprays, powders, pastes, slow-release transdermal patches, aerosols forasthma, and suppositories for application to rectal, vaginal, nasal ororal mucosa.

Thickening agents, emollients, and stabilizers can be used to preparetopical compositions. Examples of thickening agents include petrolatum,beeswax, xanthan gum, or polyethylene glycol, humectants such assorbitol, emollients such as mineral oil, lanolin and its derivatives,or squalene. A number of solutions and ointments are commerciallyavailable.

Natural or artificial flavorings or sweeteners can be added to enhancethe taste of topical preparations applied for local effect to mucosalsurfaces. Inert dyes or colors can be added, particularly in the case ofpreparations designed for application to oral mucosal surfaces.

The active compounds can be prepared with carriers that protect thecompound against rapid release, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

The active compound can also be administered through a transdermalpatch. Methods for preparing transdermal patches are known to thoseskilled in the art. For example, see Brown, L., and Langer, R.,Transdermal Delivery of Drugs, Annual Review of Medicine, 39:221-229(1988), incorporated herein by reference.

In another embodiment, the active compounds are prepared with carriersthat will protect the compound against rapid elimination from the body,such as a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc.

Liposomal suspensions may also be pharmaceutically acceptable carriers.These may be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811 (which isincorporated herein by reference in its entirety). For example, liposomeformulations may be prepared by dissolving appropriate lipid(s) (such asstearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline,arachadoyl phosphatidyl choline, and cholesterol) in an inorganicsolvent that is then evaporated, leaving behind a thin film of driedlipid on the surface of the container. An aqueous solution of the activecompound or its monophosphate, diphosphate, and/or triphosphatederivatives are then introduced into the container. The container isthen swirled by hand to free lipid material from the sides of thecontainer and to disperse lipid aggregates, thereby forming theliposomal suspension.

Modifications and variations of the present invention will be obvious tothose skilled in the art from the foregoing detailed description of theinvention. Such modifications and variations are intended to come withinthe scope of the appended claims.

We claim:
 1. A method for supressing the expression of VCAM-1 comprisingadministering an effective amount of a substance that prevents orminimizes the oxidation of a polyunsaturated fatty acid.
 2. The methodof claim 1, wherein the polyunsaturated acid is selected from the groupconsisting of oxidized linoleic (C₁₈ Δ⁹,12), linolenic (C₁₈ Δ⁶,9,12),arachidonic (C₂₀ Δ⁵,8,11,14) and eicosatrienoic (C₂₀ Δ⁸,11,14).